DE1792403A1 - Process for the production of L-lysine - Google Patents

Description

The present invention relates to a process for the production of Ir-lysine, in particular by fermentation. Specifically, the invention relates to a process for Production of L-Iysine. by fermentation with use of microorganisms with novel nutrient requirements nits

According to the previous state of the art belonged to. the microorganisms used to produce L-lysine became ,, different mutants, the homoser, Threonine. + M & thionini, threonine + cystathionine or threonine: + Need homocysteine for their growth (UBA patent specification 2 979 439) ·· The plus sign (+) as used in this specification indicates those cases in which microorganisms simultaneously require two or more types of amino acids for their growth - '

One of the disadvantages of the above fermentation processes according to the prior art was that the use of the mutant microorganisms mentioned an inappropriately, gave satisfactorily low yields of L-lysine, especially with respect to the starting material in the Amount of sugar used in nutrient medium. In addition, these processes are relative in terms of the raw material used expensive to implement, wefl. relatively large

■ p '* Amounts of amino acids or nutrients that

included, to be added to the Zuchtungsmediumi

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had to in order to have an efficient fermentation process.

The aim of the present invention is an improved method for the production of L-lysine by fermentation, which the disadvantages and deficiencies of the previous Procedure turns off.

Another object of the invention is a process for the production of L-lysine by fermentation, which carried out in an effective and relatively simple manner can be.

Another object of the invention is a process for the production of L-lysine by fermentation, which advantageously carried out on an industrial scale at low costs with high product yields v / can earth »

Another aim of the invention is the production of L-Ly s in *.

These and other objects and advantages of the present invention will be apparent to those skilled in the art from the following Description and the claims

To become a more advantageous process for the production of L-lysine by fermentation on an industrial scale create, was within the scope of the present invention Ability of many different mutant strains to

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L-lysine production is examined. In particular, it was found that Cornynebacterium glutamioum M-9ol ATCO 13287 and ATCC 14296 are extremely suitable for the production of L-lysine by fermentation (cf. US Pat. No. 2,979,439). The microbiological properties of Cornynebacterium glutamicum were first described in in U.S. Patent 3,03,925,;. £ & .. which the microorganism was identified as Micrococcus glutamiaus, and also in a recent study by Kinoshita et al from the microbiological and taxonomic point of view. This study was published in "Amino Acid", Vol. 2, 42 (196o) and "Recent progress in microbiology ¹¹ , Vol. 8" 334 (1962) and showed that the microorganism is correctly classified as Cornynebacterium was designated as Cornynebacterium glutamicum. These strains, which are the starting strains according to the invention, are mutants with nutritional requirement properties for homoserine * threonine + methionine, threonine + cystathionine or threonine. + homocystine or threonine for their growth »By irradiating these starting strains with UV According to the invention, radiation showed that special mutant strains are obtained which have both the nutritional requirements described for the parent strains and also a nutritional requirement for leucine, histidine or isoleucine + valine]

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exhibit. These new mutant strains "have an even greater ability to produce L-lysine than the original strains. Specifically, the yield based on the initial amount of sugar and the L-lysine concentration in the culture fluid obtained by using the novel mutant strains increased to around Io - 25 * f ₀

The novel mutant strains of the invention are:

Cornynebacterium glutamicum M-9ol-2347 ATCC 21253 _r Corny neb act er ium glutamicum M-9ol-2279 A (DCC 21254, Cornynebacterium glutamicum M-9ol-2234 ITCC 21255, Cornynebacterium glutamicum KY 9494 ATCCf 21299 and Cornynebacterium glutamicum 21399

Table 1 shows the nutritional requirements of the parent strains and the novel strains used according to the invention are summarized.

Tci.belie I microorganism

Cornynebacterium glutamicum M-90I (parent strain) ATCC 13287

Nutrient needs

C homoserine) or

(Threonine + methionine) or

(Threonine + cystathionine) or
(Threonine + homocysteine)

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Table (continued)

Microorganism

Oornynebacterium glutamicum M-90I-N0 ·. 2547 ATCC 21253

Cornynebacterium glutamicum

M-y01-No ₀ 2279 ATÖc 21254

Cornynebacterium glutamicum M-90I-N0. 2234 ATCC 21255 Nutrient Needs (Homoserine + Leucine or

(Threonine. +, _T ,. ^ Homocysteine) + ^leucine

^ Homoserine + histidine or

(Homoserine) +

(Threonine +
Methionine) ⁺ .

(Threonine. +
CystatMonin)

(Threonine +
Homocysteine)

Isoleucine + ¥ aline or ■

Isoleucine + valine or

+ Isoleucine ■ + valine or

+ Isoleucine + valine

Cornynebacterium glutamicum ATCC Ϊ4296 (parent strain)

Cornynebaoterium glutamicum KY 9494 ATCC 2129?

C ornynebacterium glutamicum KY 10092 ATCC 21300 threonine

(Threonine) + histidine

(Threonine) + leucine

Of course, the mutants are with the above dye needs Microorganisms not in the presence

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only the amino acids required by the parent strains grow, but only "with the addition of the amino acids that correspond to their special nutritional requirements, grow to the medium. These mutants are novel, previously unknown L-lysine-producing microorganisms When performing an L-lysine fermentation using the mutants of the invention are the composition of the basic medium and the culture conditions practically the same as commonly used and, for example, in U.S. Patent 2,979,439 are described. Either a natural or a synthetic nutrient medium is suitable, as long as it is suitable for the Growth of the specific microorganism used contains necessary nutrients · Such nutrients are in the Technique known and include substances such as a carbon source, a nitrogen source, inorganic compounds and the like in appropriate amounts to be utilized by the microorganism used. So come as carbon swell, for example, carbohydrates such as glucose, fructose, maltose, sucrose, starch, starch hydrocysates, molasses etc. or any other suitable carbon source such as glycerine, sorbitol, and also organic acids such as vinegar acid, etc. in question. These substances can either be used individually or used in mixtures of two or more

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«As a nitrogen source can be different types of inorganic or organic salts or compounds are used, such as ammonium chloride, ammonium sulfate, Ammonium nitrate, ammonium acetate. Ammonium phosphate, etc., or natural nitrogenous substances such as Corn steep liquor, yeast extract, meat extract, peptone, fish meal, BouLlon, oasein hydrolyzate, ßasamino acid, Pressed fish juices, rice bran extract, etc., These substances too can be used individually or in mixtures of two or more. Inorganic compounds contributing to the growth medium can be added are e.g. magnesium sulfate, sodium phosphate, potassium dihydrogen phosphate, potassium monohydrogen phosphate, Iron sulfate, manganese sulfate, manganese chloride, calcium chloride, calcium carbonate, sodium chloride etc. However, when raw materials such as waste molasses are used, the larger amounts of inorganic contain substances, the fermentation can be carried out without adding additional inorganic substances to the medium will.

In addition, it is responsible for the production of L-iysin Naturally necessary, the corresponding amino acids required for the growth of the microorganisms as nutrient substances to be added to the growth medium. One can use the amino acids per se or mixed, different types of Solutions containing amino acids made from natural

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Substances are obtained, use. In general, however, preference is given to various protein hydrolysates, like e.g. "Mieki" (trade name for a gluten or soy- Bean meal hydrolyzate, from which the glutamic acid is removed has been), microbial hydrolysates, soybean cake hydrolysates, Wheat gluten cake hydrolyzate and the like. Used. As shown in Table 2, these are required Quantities considerably lower than those required when using the original strains. This is one of the great features of the invention.

Nutrient substance

admitted
Amount (g./dl.)

Table 2

amount of L-lysine produced (mg./ml.)

M-9ol

M-901- M-9ol- M-9ol-

(Exit No »2234 No» 2279 No »2347

strain) ATCG ATCC ATCC

ATCC 21255 21254 21253 13287

Dried 2 • 0 37 .4 48.6 50.9 48.6 nice Wheat- 3 • 0 4o ►3 52.1 49.5 52.1 gluten- 4th • 0 44 .1 49-9 47.9 49.9 cake- 5 .0 43 .4 48.7 47.5 48.7 hydrolyte s at 1 -5 38 .5 51.4 49.2 5ο 4 Mieki 2 .0 42 .4 48.1 46.0 45.2

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Note II Composition of the basic medium: 18 g / dl

Molasses (as glucose) and 4 g / dl ammonium sulphate

Note 2 »Cultivation method: 3 1 of the cultivation medium are placed in a 5 l jar fermenter and sterilized. The strains given above are inoculated and at a pH of 7, o, a temperature of 3o ° 0, aeration cultured at 3 l / min and a speed of 400 rpm for 70 hours

The cultivation is carried out under aerobic conditions, such as a.B. aerobic shaking of the culture or by stirring a submerged culture, at a temperature of about 24 to 37 ° G, preferably about 30 ° 0, and a pEHA value of about 5-8.5, preferably carried out about 7, ο. Subject 2 to 5 days of the Growing under these conditions, substantial amounts of Ir-lysine are found to be enriched in the culture fluid obtained.

After the fermentation is complete, the Ir-lysine can be removed from the culture liquid with the usual means, such as by treatment with an ion exchange resin, extraction be separated with solvents, precipitation with metal salts, chromatography or the like. Am an essential feature of the invention is that almost no Ir-valine

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arises as a by-product, thus eliminating one of the problems which when using the original strains to produce L-lysine. This advantage is particularly evident when using the mutant strains Cornynebacterium glutamicum ATCC 21255 (a mutant that Isoleucine + VaIin-needy) and Cornynebacterium glutamicum ATCC 21253 (a leucine-requiring mutant). The cleaning process is considerably simplified in this way .

The following examples are intended to illustrate, but not limit, the invention. Palis not stated otherwise, The percentages relate to the weight per liter of water.

example 1

A Einsaatkulturflüssigkeit is prepared by 2oo ml of a culture medium containing D 5 g / dl waste molasses (as glucose), o, o5 g / dl of dipotassium hydrogenphosphate, ^o "o5 g / dl of dipotassium hydrogenphosphate, o, 3 g / dl of urea and 4 / dl Mieki, in a 500 ml Erlenmeyer flask. Cornynebacterium glutamicum M-9ol ~ 2234 ATCC 21255 is inoculated into the flask. Cultivation is then carried out with aerobic shaking for 24 hours at 30 ° C.

3 1 of a growing medium containing 18 g / dl waste

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Molasses (as glucose), 4 g / dl ammonium sulfate and 1.5 d / g Mieki are placed in a 5 liter jar fermenter and sterilized. Then 500 ml of the above seed culture liquid are inoculated into the fermentation medium and cultivation is started. Aeration of about 3 l / min and a stirring speed of about 400 rpm and a temperature of 30 ° C. are maintained during the cultivation. The pH-value is adjusted to about 6.8-7.0 with ammonia water. After 70 hours of cultivation, the concentration of L-lysine in the culture liquid obtained is 48.7 mg / ml (as hydrochloride) and the yield, based on the amount of sugar, is 27%. The concentration of the by-produced L-valine is less than o, 3 mg / ml. ^ When the growth in the same w ay, but performs, using the parent strain Cornynebacterium glutamicum ATCC 13287 as a control experiment, the concentrations are of produced L-lysine or L-valine 37.2 mg / ml or * 1.8 mg / ml *

After the cultivation is complete, the culture liquid is filtered and treated with a strongly acidic ion exchange resin according to the usual procedures. The drainage solution is then adjusted to a pH value of 5.ο and concentrated in order to bring about crystallization. ^{1 As a} result, 120 g of crude crystals of

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Obtained Ir-Iysine hydrochloride.

Example 2

3 liters of a culture medium, contain 16 g / dl sucrose, 0.15 g / dl potassium hydrogen phosphate, 0.05 g / dl dipotassium hydrogen phosphate, 4. g / dl ammonium sulfate, 0.05 g / dl magnesium sulfate, 0.02 g / dl Manganese sulphate, o, oo2 g / dl iron sulphate, 3o / ug / 1 biotin, 12ο / Ug / ml · methionine, 300 / ug / ml threonine, 500 xig / ml leucine and 100 axg / aüL cystine, are in a 5 1 -Jar fermenter 'introduced and sterilized · 5oo. ml of the seed culture liquid of Cornynebacterium glutamieum M-901-2347 ATCO 21253 · which has been previously obtained by culturing in the same manner as in Example 1 is inoculated into the fermentation medium ο The cultivation is then carried out under the same conditions as in Example 1 »

For 70 hours of cultivation, the concentration of L-lysine in the culture liquid obtained is 54.6 mg / ml (as hydrochloride). The yield, based on the amount of sugar, is 34.2 <fo. The amount of Ir-valine produced as a by-product in the medium is less than 0.5 mg / ml. If the cultivation is carried out in a control experiment in the same way as has been described above, but with the modification that the starting strain Oornynebacterium glutamieum ATCC 13287 as

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Microorganism is used, and that no leucine is contained in the starting fermentation medium, performs, the obtained concentration of L-Ly sin is 42.1 mg / mlβ 3XLe amount of the control digestion. as a by-product L-valine produced is 1.7 mg / kl.

Example 3

Cultivation is carried out in the same manner as in Example 1 except that oornynebacterium glutamicum M-9ol-2279 ATOO 21254 ale lineal strain is used. As a result, a culture liquid is obtained which contains 50.7 mg / ml L-lysine (as hydrochloride) « The yield based on the amount of sugar used in the medium is 28.1 #.

Example 4

The cultivation is carried out in the same manner as in Example 1, with the modification that öornynebac- terium glutamicum KY 9494 AiDOO 21299 is used as a linseed strain. As a result, a culture liquid is obtained which contains 53 »2 mg / ml L-lysine (as hydrochloride)»

Example 5

The cultivation is carried out in the same way as in Example 1, with the modification that öornyneba «- terium glutamieum Κγ Ioo92 ATOO 213oo used as a strain will. As a result, it becomes a culture liquid

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which contains 51.6 mg / ml L-Ijysin (as hydrochloride)

From this description of the invention it appears that it can be varied in many respects Variations are not to be regarded as deviations from the basic idea and scope of the invention, but all such modifications are intended to come within the scope of the following claims.

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Claims (1)

Patent to sayings Ια process for the production of I-lysine by fermentation, characterized in that a mutant strain of Corny ne.bacterium glutamicum, which is responsible for its growth except for the nutritional needs of the parent strains ATCC 13287 and ATCC 14296 nor leucine, histidine or isoleucine and valine required, cultured under aerobic conditions in an aqueous Uährmedium, which one for the Growth of the microorganisms sufficient amount of homoserine, threonine and methionine, threonine and cystathionine. Threonine and homocysteine or threonine and leucine, histidine or contains isoleucine and valine that L-lysine can be converted into enriches the culture liquid obtained and gains from it 2, method according to claim 1, characterized in, that the amino acids required for growth are present in the medium as part of a protein hydrolyzate are· 3. The method according to claim 1, characterized in that the cultivation is carried out at a temperature of about 24 to 37 ⁰ C and a pH of about 5 to 8.5. 4β process for the production of L-lysine by fermentation, characterized in that one has a micro COPY 109887/0660 Organism that Cornynebacterium glutamicum ATCO 21253 Cornynebacterium glutamicum ATCC 21254 Cornynebacterium glutamicum ATCC 21255 or Cornynebacterium glutamicum ATCC 21299 or Cornynebacterium glutamicum ATCC 213oo can be, under aerobic conditions in an aqueous Culture medium grows, which one for the growth of microorganisms adequate amount of homoserine, threonine and methionine, threonine and cystathionine, threonine and homocyteine or contains threonine and leucine, histidine or isoleucine and valine, that Ir-lysine is contained in the culture liquid obtained enriches and gains from it » 5 »Method according to claim 4, characterized in that that the amino acids required for growth in the medium as part of a natural substance available· 6 .. The method according to claim 5 »characterized in that that this natural substance is a protein hydrolyzate. 7 · Method according to claim 5 »characterized in that the natural substance Mieki (as in the description defined), a microbial hydrolyzate, a soybean cake hydrolyzate or a white gluten cake 109887/0660 COPY hydrolyzate is «, 8c Process according to Claim 4, characterized in that the cultivation is carried out at a temperature of approximately 24 to 37 ^° C and a pH of approximately 5 to 8.5 Z 847
Calibration: P. 109887/0660